Aromatic derivatives and iron complexes thereof for the use as normalizing agents of the iron level

ABSTRACT

The present invention is directed to aromatic derivatives of formula (I), wherein: R 1  is (a), wherein R 3  and R 4  are selected from between H and OH, provided that R 3  and R 4  are not simultaneously H, and R 2  is H; or R 1  and R 2  taken together, are (b), wherein R 5  is selected from between CH 3  and (CH 2 ) 5 OH, to the iron complexes thereof, and to their use for the preparation of pharmaceutical compositions for the normalization of iron level.

This application is the National Stage of International Application No.PCT/EP99/08222, filed on Oct. 29, 1999.

FIELD OF THE INVENTION

The present invention relates to aromatic derivatives of formula (I)hereinafter reported and to iron complexes thereof, for the preparationof pharmaceutical compositions useful as normalising agents of the ironlevel.

STATE OF THE ART

Iron overload in the body, due to major defects in iron metabolism, suchas genetic hemochromatosis and thalassemia, or due to minor causes, suchas for example transfusions for hemolytic anemia, can bring thephysiological defense reaction to saturation, thus having a toxiceffect.

Such an effect is due to the expansion of the “Free Iron Pool”, whichinduces the production of oxygen active species responsible for theinactivation of cellular enzymes, for modifications in the membranelipids, for DNA alterations, etc. (E. Cadenas, Ann. Rev. Biochem., 1989,58: 79-110).

Therefore the consequences of the “Free Iron Pool” expansion can rangefrom genotoxicity of the metal which causes the develop of tumours, tovarious organ pathologies, such as cirrhosis, diabetes, cardiopathy,hypogonadism and arthropathy.

Even in absence of an increase of the total iron in the body, anexpansion of “Free Iron Pool” may happen, in association with manypathologic conditions, as for example neurodegenerative pathologies (M.Gassen et al., Pharmacol. Toxicol., 1997, 80(4): 159-166),atherosclerosis (A. J. Matthews et al., J. Surg. Res., 1997, 73(1):35-40), ischaemic cardiopathy (T. P. Tuomainen et al., Circulation,1998, 97(15): 1461-1466), chronic diseases of intestine (R. A. Floyd etal., Dig. Dis. Sci., October 1996, 41(10): 2078-86), and may cause thebeginning or the development of the pathobiological event.

A strict correlation between alterations in the iron methabolism and HIVinfections (J. R. Boelaert et al., Infect. Agents Dis., January 1996,5(1): 36-46), as well as a connection between cardiotoxicity induced byiron and methabolism of antineoplastic agents in persons subjected toantitumoural therapy (G. Minotti et al., J. Clin. Invest., April 1995,95(4): 1595-605), has been recently observed.

Moreover, it has been proved that the presence of “Free Iron” has apredominant role in the reactions bearing to the formation of freeradicals and to the propagation of the damages due to free radicals, towhich are associated for example inflammatory pathologies, such asarthritis and pyelonephritis (R. Gupta et al., Comp. Immunol. Microbiol.Infect. Dis., 1997, 20(4): 299-307), and senescence (M. C. Corti et al.,Am. J. Cardiol., 1997, 79(2): 120-127).

At present, it is known the use of iron chelating agents in thetreatment of systemic iron overload, associated to pathologies such asthalassemia or hemochromatosis.

The compounds used in such treatments show several drawbacks. Forexample, desferrioxamine, one of the substances more frequently used inthis type of treatments, besides iron it binds many other importantmetals, such as aluminum, it is a very expensive product, it cannot beadministered by oral route, and it has a half-life so short thatcontinuous administration by subdermal infusion is necessary (R. J.Rothman et al., Mol. Pharmacol., 1992, 42: 703-710).

Other medicaments are known containing complexing agents of iron, suchas deferiprone and derivatives thereof, showing a great bioavailability,but also a high toxicity caused just by their bioavailability, whichbring for example to the onset of agranulocythemia, epathic fibrosis,etc. (N. F. Olivieri et al., N. Engl. J. Med., 1998, 339(7): 417-423).

Moreover, the medicaments, used up to now, have a dramatic effect on thecellular methabolism of iron, on which they produce a deep unbalance: asa matter of fact, they recall a great quantity of metal from thedeposits of the organism and therefore they can be administered neitherin the precox phases nor for sake of prophilaxys during hemochromatosisor thalassemia, nor in conditions in which the expansion of “Free IronPool” should be contrasted avoiding a variation in the quantity of thefunctional iron or of the iron stored in deposits.

It is therefore deeply felt the need of new compounds suitable forpreparing pharmaceutical compositions, able to act as chelating agentsof the total iron as well as of the “Free Iron Pool”, by removing itfrom cells and eliminating the resulting complex in urine.

It is felt as much the need of new pharmaceutical compositions for thetreatment of pathologies to which a lack of iron in the body isassociated.

SUMMARY OF THE INVENTION

Now the Applicant has surprisingly found that aromatic derivatives offormula (I):

wherein:

R₁ is

 wherein R₃ and R₄ are selected from between H and OH, provided that R₃and R₄ are not simultaneously H, and

R₂ is H; or

R₁ and R₂, taken together, are

 wherein R₅ is selected from between CH₃ and (CH₂)₅OH,

are able to bind stably the iron, in particular Fe(III).

Said compounds of formula (I) are suitable for the preparation ofpharmaceutical compositions for oral administration, and show a highabsorption and a high ability to permeate the biological membranes, inthe form of free ligands as well as in the form of iron complexes.

Said pharmaceutical compositions are therefore useful for the treatmentof all the pathological conditions related to an iron overload, in thecase of an increase of the total iron as well as in the case of arelative increase in the iron level due to the expansion of the “FreeIron Pool”.

The iron complexes of the formula (I) compounds are also useful for thepreparation of pharmaceutical compositions to be used in the treatmentof pathologies related to a deficiency of iron, in the acute phase aswell as for the chronic pathology.

It is therefore one object of the present invention the compounds offormula (I) for the preparation of pharmaceutical compositions for theuse, in particular, as iron chelating agents, and the complexes thereofuseful as iron release agents for hypoferremia.

Further object of the present invention are the compounds of formula (I)wherein

R₁ is

 wherein R₃ is OH and R₄ is H, OH, and R₂ is H; or R₁ and R₂, takentogether, are

 wherein R₅ is (CH₂)₅OH.

Features and advantages of compounds of formula (I) as iron chelatingagents and of the iron complexes thereof according to the presentinvention will be illustrated in detail in the following description.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to compounds having the formula (I) abovereported for the preparation of pharmaceutical compounds, in particularof pharmaceutical compounds for the use as iron chelating agents. Theiron complexes of the present formula (I) compounds have therapeuticapplication too, as agents for the iron release.

The present formula (I) compounds, which can be easily preparedaccording to processes known in the art, show a high affinity for iron,in particular for Fe(III), giving stable complexes of such metalpresenting different metal/ligand ratios. Concerning the preparation ofsaid complexes, they are tipically prepared by reacting a compound offormula (I) with Fe(III) ions deriving from a ferric salt selected fromthe group consisting of inorganic salts of Fe(III), preferablyperchlorate, chloride and sulphate, and more preferably perchlorate.

Said reaction is carried out in a solvent selected from the groupconsisting of acetone, chloroform, dichloromethane and aqueous solutionscontaining from 5 to 10% of dimethylsulphoxide, preferably in acetone.

Chelation experiments of iron have been carried out with the presentformula (I) compounds wherein R₁ and R₂, taken together, are

wherein R₅ is selected from between CH₃ and (CH₂)₅OH, and it has beenfound that they form a Fe(III) complex having a molar ratio ligand:metalequal to 2:1.

The preparation of said complex is preferably carried out at roomtemperature by reacting a compound of formula (I) with ferricperchlorate in acetone.

The stability constant of the Fe (III) complex with the compound offormula (I) wherein R₁ and R₂, taken together, are

wherein R₅ is CH₃, has been measured by voltammetry with a Pt electrodeas the working electrode and with a calomel electrode as the referenceelectrode, obtaining a stability constant equal to 3.0×10¹⁰.

Analogous chelation experiments and measurements of the stabilityconstants of the present complexes have been carried out for the otherformula (I) compounds, obtaining analogous results.

The ability of the present formula (I) compounds and of iron complexesthereof for permeating the biologic membranes has been investigated bypartition tests between n-ottanol and an aqueous solution of saidcompounds, which showed their greater affinity for the organic solvent.

In particular, partition experiments have been carried out betweenn-ottanol and an aqueous solution 20 mM oftris(hydroxymethyl)aminomethane chlorohydrate brought to pH=7.4 byadding sodium phosphate, obtaining values of the ripartitioncoefficient, expressed as the ratio between the concentration of thecompound in the organic phase and that in the aqueous phase, tipicallyhigher than 20, for the present formula (I) compounds as well as for theFe (III) complexes thereof.

The compounds of formula (I) and iron complexes thereof according to thepresent invention can be formulated with pharmaceutically acceptableexcipients and/or diluents, with the aim of preparing pharmaceuticalcompositions for the treatment of pathologies characterised by anoverload or respectively by a lack of iron in the body.

For example, dimethylsulphoxide can be used as the diluent, and examplesof excipients of possible use are methylcellulose, β-cyclodextrins andpolyethyleneglycol.

The concentration of the present formula (I) compound or that of thecorresponding iron complexes in the pharmaceutical compositions rangesbetween 0.4 and 0.8% by weight with respect to the total weight of thecomposition.

Said compositions comprising the formula (I) compounds are efficient asiron chelating agents in the treatment of the systemic ironoverload,such as in the case of thalassemia and hemochromatosis, as well as inthe treatment of the expansion of the “Free Iron Pool”.

The pharmaceutical compositions comprising the formula (I) compoundsaccording to the present invention are therefore useful in the treatmentof several pathologies to which an iron overload in the body isassociated, comprising hemochromatosis, thalassemia, anemia associatedto iron overload and conditions associated to secondary siderosis,neurodegenerative pathologies, such as Parkinson's disease andAlzheimer's disease, ischaemic cardiopathy, inflammatory chronicpathologies, such as arthritis, pyelonephritis, and inflammatory chronicdiseases of the intestine, alterations in the metabolism of ironassociated to HIV infections, cardiotoxicity due to doxorubicin andanthracyclines, atherosclerosis and senescence.

The pharmaceutical compositions comprising the iron complexes of thepresent formula (I) compounds are useful in the treatment of thepathologies to which a lack of iron in the body is associated,comprising sideropenic anemia in the acute phase as well as in thechronic form.

The following examples are given to provide non-limiting illustrationsof the present invention.

EXAMPLE 1

Preparation of 3-hydroxy-2-methyl-4H-1-benzopyran-4-one (Compound offormula (I) wherein R₁ and R₂, taken together, are

wherein R₅ is CH₃)

2-hydroxyacetophenone (20 g) is dissolved in ethyl acetate (200 ml).Sodium powder (16 g) is gradually added in 15-20 minutes, so to maintaina reasonable reflux. The mixture is refluxed for 1 hour, then it isallowed to cool. Grinded ice (200 g) is then added until precipitationof sodium acetylacetophenone, which is then isolated by filtration undervacuum. The solid product is then crystallised with acetic acid 40% (200ml), obtaining acetyl acetophenone (19.6 g) as pulverulent crystals(m.p.=95° C.; yield=75%).

Concentrated sulphuric acid (30 g) is added to acetyl acetophenone (15g) obtained as above described. After cooling at room temperature, wateris added until precipitation of a white powder, which is isolated byfiltration under vacuum and washed with cold water.2-methyl-4H-1-benzopyran-4-one (10 g) is obtained (m.p.=71° C.;yield=74%).

Pd on CaCO₃ (1.6 g) is suspended in benzene (15 ml) in a suitablehydrogenation flask. The activation of the catalysator occurs byabsorption of H₂ at room temperature, then2-methyl-4H-1-benzopyran-4-one (1.6 g) dissolved in benzene (15 ml) isadded. The catalysator is allowed to absorb ca. 200 ml of H₂ at roomtemperature and pressure. After filtration, benzene is evaporatedobtaining a pale yellow oleous liquid, to which a little volume of themixture petroleum ether:ethylic ether 1:1 is added. The obtainedsolution is purified by chromatography with a silica gel column (600-200mesh) with the above mixture as the eluent. 0.9 g of the purified anddried product are obtained (yield=60%); said product is a colourless oilwhich is resulted 2,3-dihydro-2-methyl-4H-1-benzopyran-4-one.

2,3-dihydro-2-methyl-4H-1-benzopyran-4-one (1.62 g) is dissolved inethanol 95% (50 ml) and the solution is refluxed under stirring. In10-15 minutes isoamylnitrite (8 ml) and concentrated HCl (40 ml) areadded drop by drop. Heating and stirring are stopped, and the resultingmixture is allowed to stand ca. 2 hours. Then water (200 ml) is addeduntil precipitation of a white solid. The solvent is evaporated,obtaining a solid product which is dissolved in a little volume ofchloroform and purified on silica gel in equilibrium with the samesolvent. The purified product contains a low percentage of yellowimpurities which can be completely eliminated by crystallisation inacetone. The residual solvent is then eliminated, obtaining 0.97 g of3-hydroxy-2-methyl-4H-1-benzopyran-4-one (colourless crystals;m.p.=179-180° C.; IR: 1632, 1666 e 3301 cm⁻¹; yield=51%).

EXAMPLE 2

Preparation of 3-hydroxy-2-(5-hydroxypenthyl)-4H-1-benzopyran-4-one(Compound of formula (I) wherein R₁ and R₂, taken together, are

wherein R₅ is (CH₂)₅OH)

2-hydroxyacetophenone (13.6 g) and toluene (200 ml) are introduced in areactor. Granulated sodium (4.4 g) is gradually added under stirring soto maintain a moderate reflux. Once the reaction is finished, ethyl6-hydroxyesanoate (28 g) is added, and the mixture is allowed to reactunder stirring and under a moderate reflux for 2 hours; the productformation is controlled by TLC with the mixture diethyl ether:petroleumether 9:1. After cooling, filtration under vacuum is carried out and thesolid product is then washed with diethyl ether.

The solid product is then redissolved in acetic acid 40% (100 ml), andto the so obtained solution NaHCO₃ is gradually added until neutrality.The solution is then poured in a separatory funnel and three extractionswith diethyl ether are made (each with 100 ml of the solvent). Theethereal phases from each extraction are collected together and dried byaddition of anhydrous Na₂SO₄: after the drying agent's addition thesuspension is stirred for 5 minutes, then filtered; the solvent isevaporated, obtaining an oil which is purified by chromatography with asilica gel column and the mixture diethyl ether:petroleum ether 9:1 asthe eluent. 1-(2-hydroxyphenyl)-1,3-[3-(5-hydroxypentyl)]butandione (8g) is obtained.

96% H₂SO₄ (16 g) are added to the obtained product (8 g) and the mixtureis caused to react under stirring for few minutes. After cooling at roomtemperature, water is added, then NaHCO₃ is gradually added in order toneutralise sulphuric acid. Three extractions with diethyl ether (150 ml)are carried out. The three resulting ethereal phases are collected anddried by addition of anhydrous Na₂SO₄. After filtration and evaporationof the solvent an oil is obtained which is then purified bychromatography with a silica gel column and the mixture ethylicether:acetone 80:20 as the eluent. 0.74 g of2-(5-hydroxypentyl)-4H-1-benzopyran-4-one are obtained.

Pd on CaCO₃ (0.74 g) are suspended with benzene in a suitablehydrogenation flask; the catalysator is allowed to absorb H₂ at roompression and temperature. Once the catalysator stops to absorb H₂,2-(5hydroxypentyl)-4H-1-benzopyran-4-one (0.74 g) dissolved in benzeneis added, and hydrogenation is carried out: ca. 100 ml of H₂ at roompression and temperature are absorbed. After ca. 24 hours a filtrationis carried out, and benzene is evaporated obtaining an oil to which asmall volume of diethyl ether is added. The purification of this productis carried out by chromatography with a silica gel column and diethylether as the eluent.2,3-dihydro-2-(5-hydroxypentyl)-4H-1-benzopyran-4-one (0.23 g) isobtained.

2,3-dihydro-2-(5-hydroxypentyl)-4H-1-benzopyran-4-one (0.23 g) isdissolved in ethanol 95% (5 ml), and the solution is refluxed understirring. HCl (6.2 ml) and isoamylnitrite (1.2 ml) are then added dropby drop. Stirring and heating are then stopped and the reaction mixtureis allowed to stand for ca. 1 hour. The solvent is evaporated, water isthen added and the product extracted with diethyl ether. The etherealphase is dried by addition of anhydrous Na₂SO₄. After filtration andevaporation of the solvent an oil is obtained which is purified bychromatography with silica gel column and the mixture diethylether:methanol 98:2 as the eluent.3-hydroxy-2-(5-hydroxypentyl)-4H-1-benzopyran-4-one (99.2 mg) isobtained. By crystallisation in cycloesane or diethyl ether the productin crystalline form is obtained (m.p.=111-112° C.).

EXAMPLE 3

Preparation of 3-hydroxy-2-(5-hydroxypentyl)-4H-1-benzopyran-4-one(Compound of formula (I) wherein R₁ and R₂, taken together, are

wherein R₅ is (CH₂)₅OH)

Ethyl 6-hydroxyesanoate (100 mmol) is dissolved under stirring in3,4-dihydropyrane (120 mmol); p-toluenesulphonic acid (15 mg) is thenadded and the mixture is allowed to react at room temperature for ca. 1hour. The end of the reaction is verified by TLC on silica gel and themixture petroleum ether:diethyl ether 1:1 as the eluent by using iodiumvapors as revelator. When the reaction has finished, an aqueoussolution, basic by addition of sodium carbonate, is added and accuratelystirred. The product, obtained with quantitative yield, is extractedwith diethyl ether.

Toluene (15 ml), previously distillated on sodium, is poured ongranulated sodium (300 mmol) and heated to reflux. When the reflux isreached, the heating is stopped, and ethyl 6-hydroxyesanoate (200 mmol)obtained as above described, with the hydroxylic group still protected,is added drop by drop in 90-120 minutes together with2-hydroxyacetophenone (100 mmol) in toluene (15 ml). The mixture isallowed to react for 4-5 hours, controlling the end of reaction by TLCon silica gel with the mixture petroleum ether:diethyl ether 1:1 as theeluent.

At the reaction's end ethanol is added so to eliminate the excess ofsodium, then acetic acid 7.5% (100 ml) is added, controlling that pH isca. 10.

The condensation product with the hydroxylic group still protected isextracted by carrying out three extractions with diethyl ether and twoextractions with ethyl acetate, finally obtaining the product with a 60%yield.

The so obtained product, dissolved in acetic acid (2 ml), is put into areactor, which is immersed in an oil bath, so to bring the temperatureto 130° C., and maintain it for ca. 1 hour. At first the distillation ofthe azeotrope water/acetic acid occurs, then a vacuum pump is applied inorder to promote the distillation of the remaining acetic acid.

The cyclisation is verified by TLC on silica gel with diethyl ether asthe eluent. At the end of cyclisation a mixture acetic acid:water 4:1(50 ml) is added and the temperature is brought to 50° C. The reactionmixture is maintained under stirring at 50° C. until the protectiongroup is completely removed, obtaining2-(5-hydroxypentyl)-benzopyran-4-one. The formation of this product isverified by TLC on silica gel with diethyl ether containing 2% ofmethanol as the eluent.

The obtained product is extracted with diethyl ether after the additionof water, then the solvent is evaporated and the product is purified bycolumn chromatography with diethyl ether containing 2% of methanol asthe eluent, thus obtaining pure2-(5-hydroxypentyl)-4H-1-benzopyran-4-one with a 50% yield.

Pd 5% on calcium carbonate (1 g) is put in ethanol (20 ml) andhydrogenated. When the catalysator does not absorb H₂ anymore, asolution prepared by dissolving in ethanol (40 ml)2-(5-hydroxypentyl)-4H-1-benzopyran-4-one (4 g), obtained as describedabove, is added, and hydrogenation is carried out. The mixture is causedto react for 4-5 hours, then filtered; the reaction product isessiccated and redissolved in the minimum quantity of diethyl ether forthe purification by chromatography on a silica column with diethyl etheras the eluent. 2,3-dihydro-2-(5-hydroxypentyl)-4H-1-benzopyran-4-one isthus obtained with 50% yield.

The so obtained product (1 mmol) is dissolved in ethanol 95% (5 ml), andthe solution is refluxed under stirring. HCl (6.5 ml) and isoamylnitrite(1.2 ml) are added drop by drop to this solution. Then heating andstirring are stopped, and the reaction mixture is allowed to stand for 1hour. The solvent is evaporated, water is added, making then anextraction with diethyl ether; the ethereal phase is dried by additionof anhydrous Na₂SO₄. By filtration and avaporation of the solvent3-hydroxy-2-(5-hydroxypentyl)-4H-1-benzopyran-4-one is obtained with 25%yield. This product is then purified by chromatography on silica columnwith the mixture diethyl ether:methanol 98:2 as the eluent, thusobtaining pure 3-hydroxy-2-(5-hydroxypentyl)-4H-1-benzopyran-4-one(fluorescent under a light with λ=360 nm;

¹H-NMR (CDCl₃, 200 MHz) δ from 1.5 to 1.88 ppm (multiplet), 2.5 ppm(triplet), 3.6 ppm (triplet), from 7.35 to 8.25 ppm (aromatic H); MS m/e248 M⁺).

EXAMPLE 4

Preparation of the Fe(III) complex of3-hydroxy-2-methyl-4H-1-benzopyran-4-one

3-hydroxy-2-methyl-4H-1-benzopyran-4-one (7 mg; 0.04 mmol), prepared asdescribed in Example 1, is dissolved in acetone (5 ml); to this solutionferric perchlorate (7 mg; 0.02 mmol) is added at room temperature, thusobtaining the quantitative formation of the complex chelatingagent:Fe(III) 2:1. By evaporation of acetone the complex is isolated asa dark red pulverulent residue, characterised by mass spectroscopy.

The complexation cynetics has been moreover studied by cyclicvoltammetry with a Pt electrode as the working electrode and a calomelelectrode as the reference electrode, in the following conditions:ferricperchlorate (7 mg) is dissolved in 20 ml of a NaCl solution 150 mMcontaining 5% of DMSO, and this solution is put into the voltammetriccell for the plot registration; from this plot the half-wave potentialfor the ferric salt can be deduced: (E_(½))s Fe³⁺/Fe²⁺=0.420 V.

3-hydroxy-2-methyl-4H-1-benzopyran-4-one (10.5 mg) is dissolved in DMSO(3 ml), and with the so obtained solution 4 additions are made to thevoltammetric cell, registering the cyclic voltammetric plot after everyaddition. After the first addition of 0.5 ml the plot is registered,from which the half-wave potential of the complex is obtained: (E_(½))sFe³⁺/Fe²⁺=0.120 V. Successively 0.5 ml (second addition), 1 ml (thirdaddition), 1 ml (fourth addition) of the solution of the chelating agentin DMSO are then added. After the third addition the oxidation waveFe³⁺/Fe²⁺ is disappeared, thus proving that free iron is not in thesolution after the molar ratio chelating agent:metal 2:1 is reached.

The stability constant of the complex is calculated according to thefollowing formula:${{\left( E_{1/2} \right)s} - {\left( E_{1/2} \right)c}} = {{\frac{0\text{,}059}{n}\log \quad \beta} + {\frac{0\text{,}059}{n}p\quad \log \quad c}}$

wherein (E_(½))s e (E_(½))c are the half-wave potential of the salt and,respectively, of the complex above reported, n is the number ofelectrons, β is the stability constant of the complex and c is itsconcentration, and p is the number of chelating agent molecules in thecomplex. The so calculated value of the stability constant is3.0×10_(10.)

EXAMPLE 5

Preparation of the Fe(III) complex of3-hydroxy-2-(5-hydroxypentyl)-4H-1-benzopyran-4-one

According to a procedure similar to that described in Example 4, theFe(III) complex of the3-hydroxy-2-(5-hydroxypentyl)-4H-1-benzopyran-4-one obtained as inExample 3, is prepared (MS m/e 549 M⁺). This indicates the formation ofa neutral complex having a ratio chelating agent:Fe(III) 2:1, in whichthree of the four oxydrylic groups of chelating agent are deprotonatedin the complex.

EXAMPLE 6

Partition test on 3-hydroxy-2-methyl-4H-1-benzopyran-4-one and on theiron complex thereof

The partition coefficient between n-octanol and an aqueous solution 20mM of tris(hydroxymethyl)aminomethane brought to pH=7.4 by the additionof sodium phosphate has been determined by spectrophotometry at roomtemperature for 3-hydroxy-2-methyl-4H-1-benzopyran-4-one and for therelated Fe(III) complex.

In the aqueous solution the 3-hydroxy-2-methyl-4H-1-benzopyran-4oneobtained as described in Example 1 was dissolved in the quantitysufficient to have a concentration equal to 10⁻⁴ M. 5 ml of saidsolution were centrifuged with 5 ml of n-octanol, and the two resultingphases were separated for a concentration determination on each byspectrophotometry.

The partition coefficient, expressed as the ratio between theconcentration of the compound in n-octanol and its concentration in theaqueous solution, resulted 24. In the same aqueous solution abovedescribed the Fe(III) complex obtained as in Example 4 was dissolved, inthe sufficient quantity to obtain a concentration equal to 2×10⁻⁴ M. 5ml of said solution are centrifuged with 5 ml of n-octanol, then the tworesulting phases were separated for a concentration determination oneach by spectrophotometry. A partition coefficient equal to 25 wasobtained.

EXAMPLE 7

Partition data on 3-hydroxy-2-(5-hydroxypentyl)-4H-1-benzopyran-4-oneand on the related iron complex

According to the procedure described in Example 6 partition data weredetermined between n-octanol and an aqueous solution 20 mM oftris(hydroxymethyl)aminomethane chlorhydrate brought to pH 7.4 byaddition of sodium phosphate, for3-hydroxy-2-(5-hydroxypentyl)-4H-1-benzopyran-4-one prepared as inExample 3 as well as for the related Fe(III) complex prepared as inExample 5.

In the aqueous solution3-hydroxy-2-(5-hydroxypentyl)-4H-1-benzopyran-4-one as prepared inExample 3 was dissolved so to obtain a concentration equal to 100 mM. 5ml of said solution were centrifuged with 5 ml of n-octanol, then thetwo resulting phases, the aqueous and the organic one, were separatedfor a concentration determination on each by spectrophotometry.

The same experiment was repeated by dissolving the Fe(III) complexprepared as in Example 5 in the above described aqueous solution.

In both cases the compound completely pass in the alcoholic phase.

EXAMPLE 8

In vitro tests on permeation into erythrocytes of3-hydroxy-2-methyl-4H-1-benzopyran-4-one and of the related Fe(III)complex

Solutions of 3-hydroxy-2-methyl-4H-1-benzopyran-4-one prepared as inExample 1 and the related Fe(III) complex obtained in acetone as inExample 4 were used for suitable sampling in incubation flasks. For thecomplex acetone is evaporated, and the biologic samples are then addedto the residue. Whole blood of rat deprived of leucocytes, oralternatively erythrocytes washed and resuspended in 127 mM of sodiumphosphate 50% buffer (pH=7.4) and containing 23 mM of NaCl, were addedinto the incubation flasks and incubated at 37° C. for 15 andrespectively 30 minutes.

At those times erythrocytes were separeted by centrifugation andseparately extracted together with the supernatants with 2 volumes ofethyl acetate. The related quantities of the tested substances weremeasured by spectrophotometry in ethyl acetate and compared withsuitable standards. After 30 minutes of incubation the3-hydroxy-2-methyl-4H-1-benzopyran-4-one as well as the related Fe(III)complex were found in the erythrocytes for 51% and for 49% in theaqueous medium. After 30 minutes of incubation 50% of the sedimentederythrocytes are resuspended in their fresh plasma and the remaining 50%in fresh buffer.

After a further 30 minutes incubation at 37° C. the3-hydroxy-2-methyl-4H-1-benzopyran-4-one as well as the related ironcomplex were equally distributed in the erythrocytes, previouslyoverloaded, and in the aqueous medium in which they were resuspended,thus proving that such compounds are distributed in a practicallyuniform way in the intracellular and in the extracellular environment,and a balance is reached in ca. 30 minutes as far as erythrocytes areconcerned.

EXAMPLE 9

In vitro tests on permeation into erythrocytes of3-hydroxy-2-(5-hydroxypentyl)-4H-1-benzopyran-4-one and of the relatedFe(III) complex

Erhytrocytes of rat were separated from their plasma deprived ofleucocytes. Part of the erythrocytes were resuspended in sodiumphosphate 50% buffer (pH=7.4), and another part of the erythrocytes werejoined to part of their plasma so to obtained a 50% hematocrit. To bothsuspensions were added alternatively 100 mg/l of3-hydroxy-2-(5-hydroxypentyl)-4H-benzopyran-4-one prepared as in Example3 and 50 mg/l of the related Fe(III) complex prepared as in Example 5,and the incubation was made at 37° C. After 30 minutes the erythrocyteswere separated by centrifugation and extracted separately together withsupernatants with ethyl acetate. The quantities of the tested substanceswere measured by spectrophotometry in ethyl acetate and compared tosuitable standards.

After 30 minutes incubation the 40% of3-hydroxy-2-(5-hydroxypenthyl)-4H-1-benzopyran-4-one, as well as of theferric complex thereof, were found in the erythrocytes and the remaining60% in the medium represented by plasma. When the medium is thephosphate buffer the 60% of the tested compounds were found inerythrocytes and the remaining 40% in the medium.

After sedimentation 50% of the erythrocytes containing the testedcompounds was resuspended in their plasma and the remaining 50% in freshbuffer. After a further 30 minutes incubation at 37° C. 60% of3-hydroxy-2-(5-hydroxypentyl)-4H-1-benzopyran-4-one and of the relatedcomplex were found in the plasmatic medium and the remaining 40% in theaqueous medium containing the phosphate buffer. Therefore the chelatingagent as well as the related ferric complex do permeate into cellularmembranes in both directions, and are uniformly distributed in theintracellular and in the extracellular environment.

EXAMPLE 10

In vivo tests on permeation of rat erythrocytes by3-hydroxy-2-methyl-4H-1-benzopyran-4-one

The tests are performed on male rats Sprague-Dawley weighting from 250to 300 g.

The chelating agent 3-hydroxy-2-methyl-4H-1-benzopyran-4-one prepared asin Example 1, vehiculated by 4 parts of β-cyclodextrine for each part ofchelating agent, is administered to the rats in a single oral dosecomprising 20 mg of chelating agent per Kg of body weight. At regulartime intervals, after ½ hour, 1 hour, 4 hours, 24 hours e 48 hoursstarting from administration, the presence of the chelating agent in therats is evaluated by spectrophotometry and by fluorimetry:3-hydroxy-2-methyl-4H-1-benzopyran-4-one is found already in the firsthour in erythrocytes, in brain and liver showing a maximum in the fourthhour, and in urine starting from twenthyfourth hour.

EXAMPLE 11

In vivo tests on permeation of rat erythrocytes by3-hydroxy-2-(5-hydroxypentyl)-4H-1-benzopyran-4-one

The tests are performed on male rats Sprague-Dawley weighting from 250to 300 g.

The chelating agent 3-hydroxy-2-(5-hydroxypentyl)-4H-1-benzopyran-4-oneprepared as in Example 3 is administered to the rats in a single oraldose, comprising 2% of methyl cellulose and 20 mg of chelating agent perKg of body weight. The presence of3-hydroxy-2-(5-hydroxypentyl)-4H-1-benzopyran-4-one has been revealed inurine after 12 hours, 24 hours and 48 hours from administration. When 20mg of 3-hydroxy-2-(5-hydroxypentyl)-4H-1-benzopyran-4-one per Kg of bodyweight are administered in DMSO by intraperitoneal route, its presencein plasma and erythrocytes has been revealed after 15, 30 e 60 minutesafter the treatment.

TABLE 1 Qualitative detection of3-hydroxy-2-methyl-4H-1-benzopyran-4-one in rat tissues and itsexcretion. Time after adminis- tration Plasma Erythrocytes Liver BrainUrine Faeces 1/2 hr  p(+) p(+) A a a a  1 hr p(++) p(++) p(+) p(+) a a 4 hr p(++) p(+++) p(+++) p(++) p(+) a 24 hr a a p(++) p(++) p(++) a 48hr a a a a p(+++) a

TABLE 2 Qualitative detection of3-hydroxy-2-(5-hydroxypentyl)-4H-1-benzopyran- 4-one in rat tissues andits excretion. Time after adminis- tration Plasma Erythrocytes LiverBrain Urine Faeces 1/4 hr  p(+) p(+) a a a a 1/2 hr  p(++) p(++) a a a a 1 hr p(++) p(+++) p(+) a a a  4 hr p(+++) p(+++) p(++) a a a 12 hr a ap(++) p(+) p(+) a 24 hr a a p(+++) p(+) p(+++) a 48 hr a a a a p(+++) ap = present; a = absent. Plus sign indicates a semi-quantitative score.

What is claimed is:
 1. A pharmaceutical composition comprising as theactive ingredient at least one compound of formula (I)

wherein: R₁ is

 wherein R₃ and R₄ are selected from the group consisting of H and OH,provided that R₃ and R₄ are not simultaneously H, and R₂ is H; or R₁ andR₂, taken together are

 wherein R₅ is (CH₂)₅OH, or an iron complex thereof.
 2. A pharmaceuticalcomposition comprising as the active ingredient an iron complex of thecompound of formula (I) as defined in claim 1 wherein R₅ is CH₃.
 3. Thepharmaceutical composition according to claim 1, further comprisingpharmaceutically acceptable excipients and/or diluents.
 4. A compound offormula (I)

wherein R₁ is

 wherein R₃ is OH and R₄ is H, OH; and R₂ is H; or R₁ and R₂, takentogether, are

 wherein R₅ is (CH₂)₅OH.
 5. The pharmaceutical composition according toclaim 2, further comprising pharmaceutically acceptable excipientsand/or diluents.
 6. A method of treating the conditions associated withan iron overload comprising administering to an individual affected byan iron overload an effective amount of a compound of formula (I)

wherein: R₁ is

 wherein R₃ and R₄ are selected from the group consisting of H and OH,provided that R₃ and R₄ are not simultaneously H, and R₂ is H; or R₁ andR₂, taken together, are

 wherein R₅ is selected from the group consisting of CH₃ and (CH₂)₅OH.7. The method according to claim 6, wherein the conditions with which aniron overload is associated comprise hemochromatosis, thalassemia,anemia associated with iron overload and conditions associated withsecondary siderosis, neurodegenerative pathologies comprisingParkinson's disease and Alzheimer's disease, ischaemic cardiopathy,inflammatory pathologies comprising arthritis, pyelonephritis andchronic inflammatory diseases of intestine, alterate conditions in theiron metabolism associated with HIV infections, cardiotoxicity due todoxorubicin and anthracyclines, atherosclerosis and senescence.
 8. Aniron complex of the compound of formula (I)

wherein: R₁ is

 wherein R₃ and R₄ are selected from the group consisting of H and OH,provided that R₃ and R₄ are not simultaneously H, and R₂ is H; or R₁ andR₂ taken together are

 wherein R₅ is selected from the group consisting of (CH₂)₅OH and CH₃.9. The iron complex according to claim 8, wherein the molar ratio offormula (I) to iron is 2:1.
 10. The iron complex according to claim 8,wherein iron is Fe (III).
 11. A method of treating the conditions whichare associated with a lack of iron comprising administering an effectiveamount of iron complexes as defined in claim
 8. 12. The method accordingto claim 11, wherein the conditions with which a lack of iron isassociated comprise sideropenic anemia in the acute phase and chronicsideropenic anemia.